1. Field of the Invention
The present invention relates generally to instruments and methods for analyzing airborne biological particles. More particularly, the present invention relates to instruments and methods for classifying and identifying airborne biological particles in real-time based on both their size and laser-induced auto-fluorescence of their biological components.
2. Background of the Invention
Numerous airborne pathogens and allergens can be found in or introduced into an environment. These airborne particles can be naturally occurring or artificially produced. Some of these airborne particles can be dangerous, and even life threatening. For example, such airborne particles can include biological agents such as those that can be used for military purposes or as weapons of terror by terrorist organizations. To avoid widespread illness or death, there is a need for real-time detection of airborne biological particles. Early warning provided by real-time detection of such airborne biological particulates minimizes human exposure to such harmful airborne pathogens, allergens, and biological warfare agents.
For military and counter-terrorism applications, rapid detection of a biological aerosol attack is often essential for effective treatment. Table 1, for example, shows exemplary exposure levels required (in numbers of biological warfare agent particles) to produce an infectious response by inhalation if treatment is not provided in a timely manner.
TABLE 1Numbers of biological particle likely to be lethalif inhaled and not timely treatedNumber of Particles Required to Produce aBiological Warfare AgentLethal Response by InhalationBacillus anthracis spores8,000-10,000(Anthrax)Yersinia pestis  10-100(Bubonic Plague)Franciscella tularensis  10-100(Tularemia)Smallpox   1-10
Due to the low number of biological particles required for various agents to produce an infectious response, biological aerosol detection systems should be able to detect low levels of biological warfare agent aerosol concentrations. Aerosol attacks delivering, as low as, a biological particle per liter of air can still provide a life threatening level of biological warfare agents. Consequently (referring to Table 1), a major challenge for real-time detection systems is that they be capable of detecting trace levels of airborne biological agents.
Other applications also require the real-time detection of individual airborne biological particles. For example, airborne detection of organisms in pharmaceutical and biotechnology production facilities can be used to provide verification and validation that there has been no contamination to drugs or other biological compounds produced in these environments. Airborne detection of biological particles can also be used to monitor organism levels in hospitals and other critical care facilities to prevent post-operative infections and the spreading of disease in such facilities. Monitoring of airborne biological particulates at animal processing and sewage treatment facilities can be used to ensure industrial hygiene. Detection of commonly encountered pathogens in a building's HVAC system can be used for purposes of indoor air quality monitoring. Detection of pollens and other biological particulates in outdoor environments can also be used in meteorological and aerosol research applications. Thus, it can be seen that real-time detection of biological particles is required for a variety of applications, including those described above.
One method for characterizing individual airborne biological particles in real-time is measuring laser-induced auto-fluorescence of biological particles. The majority, if not all, biological particles in nature have one or more endogenous fluorophores associated with it. Such endogenous fluorophores comprise biological or biochemical components that absorb light at a particular frequency and emit fluorescence at another particular frequency. The emitted fluorescence frequency is dependent on the absorption frequency. The biological or biochemical components in different endogenous fluorophores are excited by light having different frequencies and fluoresce at different frequencies. These different fluorescence frequencies can be used as a signature to characterize and identify each biological or biochemical component in a biological particle. That is, because different biological particles are comprised of different combinations of biological or biochemical components, as well as, different concentrations of endogenous fluorophores relative to the biological particle's size, detection and analysis of fluorescing and Mie scattering characteristics can be used to detect and classify the biological particles.
These endogenous fluorophores include flavins, the coenzymes NADH and NADPH, the amino acids tryptophan and tyrosine, porphyrins. Table 2 provides a list of common endogenous fluorophores, and their corresponding absorption and fluorescence emission wavelengths.
TABLE 2Endogenous Fluorophore Absorption and Fluorescence MaximaFluorophoreAbsorption (nm)Fluorescence (nm)Tryptophan 220,280,288320-350Thyrosin220,275305Collagen300-340420-460Elastin300-340420-460NADH260,340470NADPH260,340470Flavins260,370,450530Zn-coproporphyrin411,539,575580Zn-protoporphyrin421,548,585592Uroporphyrin404,501,533,568,622624Coproporphyrin398,497,531,565,620622Protoporphyrin406,505,540,575,630633Chlorophyll a425,670685Chlorophyll b455,642660
Auto-fluorescence of individual biological particles is typically induced by exciting the biological particles with a laser. Laser-induced auto-fluorescence uses a laser to illuminate a biological particle with light having a wavelength that causes endogenous fluorophores in the biological particle to fluoresce.
To more accurately classify biological particles, their size can also be considered. Certain known biological particles have certain sizes. Particles of different sizes can contain similar endogenous fluorophores. Consequently, combining size and auto-fluorescence information can provide more accurate detection and characterization of particular biological agents.
Conventional techniques for detecting individual airborne biological particles use a two laser system. A first laser is used to determine particle size. The second laser is a UV laser that is used to provide an auto-fluorescence measurement. Particle size is determined by time of flight or Mie scattering (also called elastic scattering) measurements. The second laser is pulsed based on a triggering signal generated as a function of the particle's size. Such a system, for example, is disclosed in U.S. Pat. No. 5,999,250 to Hairston et al. (“Hairston”). Hairston discloses a two laser system for determining particle size and detecting fluorescence wherein a first laser (in the visible region) is used to determine a particle's size by a time-of-flight measurement. The particle size is then used to determine a delay (based on particle size) after which to trigger the second laser (in the UV region) as the particle passes through the second laser's beam.
U.S. Pat. No. 5,895,922 to Ho also discloses a two-laser system for determining particle size and detecting fluorescence wherein a first laser (in the visible region) is used to determine a particle's size by a time-of-flight measurement. In one embodiment of the system disclosed in Ho, the UV laser is triggered on the basis of the particle's location. In another embodiment of the system disclosed in Ho, the UV laser is continuous. In this embodiment, a window generator opens a collection window (of approximately 1 us) during which fluorescence signals from a PMT are collected.
One problem potentially encountered with such triggered systems is an increased likelihood of not detecting a certain percentage of biological particles at elevated aerosol concentrations due to triggering limitations. This is a significant issue when detecting airborne biological particles due to the fact that the percentage of biological particles in most environments is less than 0.1% of the total aerosol content.